The present invention relates to methods for the determination of a target binding pair member, such as an antigen, antibody, hapten or nucleic acid strand in a biological sample; and the invention also relates to reagents or kits used for such determination method. The method involves, in general, amplified read-outs, such as by the use of enzymes, and also involves transferring a detectable moiety into a gas phase where the detectable moiety is detected (determined either quantitatively or qualitatively).
Enzyme immunoassays are well known in which a binding reaction, such as between sample and labeled antigens and reagent antibodies, are conducted. At the conclusion of the assay, with or without a separation, an enzyme is present in a phase, either a liquid phase or a solid phase, and the concentration and/or activity of the enzyme is functionally related to the concentration (or presence) of the target binding pair member in the sample assay. Exemplary references to such enzyme immunoassays include Enzyme-Immunoassay (CRC Press 1980, E.T. Maggio ed.). Such enzyme immunoassays can generally be characterized as either heterogeneous (competitive or non-competitive) or homogeneous. These different geometries lead to different results in that, in the competitive-heterogeneous assay, higher sample concentrations of the target antigen generally lead to lower concentrations of enzyme in the solid phase after separation. In the heterogeneous non-competitive assay (e.g., a sandwich assay) higher concentrations of the target antigen or antibody generally lead to higher concentrations (and therefore activities) of enzyme on the solid phase after separation. In the homogeneous assay, the concentration of enzyme in the liquid phase after the binding reaction is unaffected; however, the activity of the enzyme is modulated by the target concentration, with some forms having lower enzyme activity with higher target antibody concentrations, but other forms having higher enzyme activities with higher target antigen concentrations. Surveys of geometries for enzyme immunoassays are contained in E.T. Maggio, Enzyme-Immunoassay (CRC Press, Inc., Boca Raton, FL 1980); T. T. Ngo et al., Mol. & Cellular Biochem., Vol. 44, pp. 3-12 (1982); C. Blake et al., Analyst, Vol. 109, pp. 533-547 (May 1984).
Assays for target polynucleotide sequences (DNA or RNA) have been described based upon the specific hydrogen bonding between purine/pyrimidine nucleotides. The most common form of these assays involves immobilizing the nucleic acid of a sample in single stranded form on a support. Thereafter a probe, containing nucleotide sequences complementary to the target strand, is applied with a label such as either an enzyme or a binding member (e.g., biotin or an antigen) pendent to the probe polynucleotide. If the enzyme is not directly attached to the probe polynucleotide, it may be indirectly attached by using a probe bearing an antigen (e.g., biotin) and subsequently contacting the support with an enzyme linked to an antibody (or, similarly, avidin or strepavidin, each of which binds to biotin). After washing the coated support to remove any unbound probebiotin conjugate or any unbound avidin-enzyme conjugate, the coated support is contacted with substrates for the enzymatic reaction. After a controlled period, either reactants or products of the enzymatic reaction may be assayed, with increased amounts of enzymatic reaction corresponding to increased concentrations of the target polynucleotide in the sample. Such methods are described generally in Falkow et al., U.S. Pat. No. 4,358,535 (1982); EPO 70,687 of Heller et al. (Standard Oil 1983) (page 6), and forms of such methods employing biotin or the like attached to the probe are described in EPO No. 63,879 of Ward et al. (Yale University 1982); EPO No. 97,373 of Rabbani et al. (Enzo Biochem, Inc. 1984); Leary et al., Proc. Nat. Acad. Sci., Vol. 80, pp. 4045-4049 (1983).
A second type of polynucleotide assay which has been described is the "sandwich" assay, somewhat similar to sandwich assays used for immunochemical analyses. In such sandwich assay two probe polynucleotides are used, each complementary to a portion of the target polynucleotide strand. The first probe polynucleotide is attached to a support. The second probe polynucleotide can be attached to either an enzyme or to biotin (which is subsequently attached to the enzyme using an avidinenzyme conjugate or the like). By simultaneous or sequential incubation, a complex is formed containing support linked by the first probe target polynucleotide strand, and the second probe (and, optionally, further through biotin and avidin) to the enzyme. After appropriate washing, such complexes should represent the only enzyme remaining coated on the support. By then introducing the reactants of the enzymatic reaction, and assaying for the products of the enzymatic reaction, enzyme concentration on the first phase (the support) can be quantified. Such enzymatic activity should be positively related to the concentration of the target nucleotide strand in the sample. See EPO No. 70,687 (Standard Oil 1983) (pages 8-10); PCT Application WO 83/01459 of Orion-Yhtma Oy (Apr. 29, 1983).
A third general type of polynucleotide assay has been described in U.S. patent application Ser. No. 607,885 of Diamond et al., entitled "Displacement Polynucleotide Assay And Method Polynucleotide Complex Reagent Therefor", assigned to Allied Corporation and Genetics Institute Inc. and filed on May 7, 1984. By virtue of the assignment of the present application to Allied Corporation, it is believed that there is sufficient common assignment of these two applications for this reference. In such systems, the probe polynucleotide, complementary to the target polynucleotide, is provided in solution or on a support, but paired through hydrogen bonding to a second polynucleotide, called the labeled polynucleotide. In some forms of that invention, the labeled polynucleotide contains either an enzyme as a label, or contains a binding determinant such as biotin as the label. In either case, when this reagent complex is contacted with sample, target polynucleotides in the sample will displace labeled polynucleotide from the reagent complex. By either conducting a separation or by the effect of the displacement on the activity of the enzyme (frequently due to steric factors) the activity and/or concentration of the enzyme in a phase (generally a liquid phase) will be positively related to the concentration of target polynucleotide in the sample.
While all of the above procedures take advantage of the amplification of an enzyme to enhance the detectable moieties compared to the target binding pair member moieties, a common problem in such assays is distinguishing such detectable moieties from the many other organic and biological materials present in the phase, particularly liquid phase, wherein the binding reaction and/or the enzymatic reaction are conducted. In the case, particularly, of fluorescence or absorption measurements, such interferences are quite common and can lead to significantly less selectivity and/or sensitivity to the assay than is dictated by the specific binding reaction or the enzymatic reaction. Nevertheless, any method proposed to overcome these interferences must offer simplicity and speed similar to that of the first two reactions in order not to unduly complicate or delay the obtainment of qualitative or quantitative results from the assay.